STATE OF THE ART The process of wound repair involves a complex interaction between cascades of serum enzymes, local action of growth factors, circulating platelets and monocytes, tissue macrophages, fibroblasts, endothelial cells, epithelial cells and local cell microenvironment.

Data from the literature have demonstrated that repair of damaged tissue and blood vessels is a problem for all types of speciality surgery and, moreover, the normal process of healing can be slowed down due to concurrent diseases such as diabetes.

The resolution of such problems has been attempted in several ways.

Although widely used, hemostatic agents for topical use consisting of modified biological materials are of limited effectiveness as sealants and are totally ineffective as binders in that they cause an inflammatory type immune response which further prolongs the healing process.

The fibrin glue obtained by the activation of fibrinogen by thrombin and the relative polymerisation of the resulting fibrin have been extensively proposed as a general solution to these problems.

For example W096/27397 describes an autologous concentrate termed"plasma buffy coat"which comprises plasma, platelets and fibrinogen at concentrations much higher than physiological ones; this concentrate is used for preparing fibrin glues in the presence of a fibrinogen activator and, in particular, thrombin. In Oral Surg. Oral Med. Oral Path. 1998,85 : 638-646, Marx and his collaborators

demonstrated that the use of platelet concentrate is an innovative method for regulating and accelerating healing processes, also bone regeneration, in that it allows a platelet gel to be obtained capable of enhancing the effects of the growth factors contained in the platelet granules, being initiators of the metabolic pathway concerned in the healing process.

According to Marx's protocol, which involves the use of a cell separator, the PC is prepared by taking a 500 ml blood sample by means of an operation involving a venous catheter. The PC is subsequently activated with calcium chloride and bovine thrombin to produce a platelet gel which can be used either with a matrix of cortical and/or medullar bone or with a synthetic bone matrix. US 5,585, 007 and WO 98/11925 refer to methods for preparing platelet gel. All the aforementioned processes for preparing platelet gel and fibrin glue propose the use of either human or bovine thrombin as activator.

Human thrombin can be prepared by recombinant DNA technology and the products obtained with this type of technique can be used only for experimental purposes, essentially in vitro.

Another procedure which enables human thrombin to be prepared uses an open system, but the final product is not sterile.

Finally there is a last alternative system, in the experimental phase, which enables human thrombin to be obtained, achieved by its isolation using special equipment.

Bovine thrombin is much more available, however its relative use is not advised due to problems connected with BSE and allergic reactions.

In international patent application W001/43787 a process is described which allows the technical problems connected with BSE to be overcome. This technique proposes the preparation of a platelet gel by activating PC with an aqueous solution containing batroxobin and a calcium salt.

In particular, the batroxobin used according to said technique, commercially available and known as Botropase, is an aqueous solution containing 1 international unit/ml of batroxobin, sodium chloride and 0.3% of phenol as preservative.

Although this technique is undoubtedly innovative with respect to known platelet gel preparation techniques involving the use of thrombin, it nevertheless presents

a drawback caused by having a cytotoxic effect, even if used at low concentrations such as those proposed in the aforesaid patent application and in particular even at 0. 5 NIH/ml.

In addition botropase at the aforementioned concentration (0.5 NIH/ml), (wherein NIH means thrombin international unit) requires considerably long activation times for precipitating PPP or PRP gel.

TECHNICAL PROBLEM The need was therefore felt for an activator able to form platelet gel, PPP gel or PRP gel without the drawbacks of the known art, while at the same time being able to accelerate the healing process and to favour cell growth and proliferation.

SUMMARY OF THE INVENTION The Applicant has surprisingly found that it is possible to obtain the aforementioned results with a gel forming activator, chosen from the group consisting of platelet gel, PPP gel and PRP gel, comprising or preferably consisting of an aqueous solution containing batroxobin and a calcium salt, characterised in that said aqueous solution contains batroxobin in essentially pure form, said aqueous solution being essentially devoid of phenols.

The Applicant has in fact surprisingly also found that the use of this type of activator, with batroxobin at concentrations of 50 BU/ml, does not cause any cytotoxic effect (1 BU (biological unit) corresponds to 0. 18NIH).

In addition the Applicant has also unexpectedly found that by using the activator according to the present invention containing the batroxobin in a substantially pure form requires shorter activation times if compared to those obtained with botropase, also in the case the former batroxobin is present at concentration decidedly lower than that obtained with botropase (see example 1).

Therefore, a further aspect of the present invention is a method for preparing platelet gel comprising the treatment of PC with the activator of the present invention.

A further aspect of the present invention is a method for preparing a PPP gel by treating PPP with the activator of the present invention.

A further aspect of the present invention is a method for preparing PRP gel comprising the treatment of PRP with the activator of the present invention.

A further aspect of the present invention consists in the kits for carrying out the aforementioned methods for preparing respectively autologous platelet PPP or PRP gel.

In particular the kits according to the present invention are of two types, namely the wound care activation kit useful for the treatment of cutaneous non healing lesions and the surgery activation kit to be used for surgical practice such as oral, maxillofacial and orthopedic surgery.

The Applicant has also found that the activator of the present invention in addition to not being cytotoxic, produces platelet gel, PPP gel or PRP gel of autologous type, allowing cells to grow and proliferate in the absence of heterologous culture media of conventional type which contain cell growth and proliferation promoters such as Foetal Calf Serum (FCS) or Foetal Bovine Serum (FBS); in addition, cell growth and proliferation, in the presence of platelet gel, PPP gel or PRP gel is definitely increased with respect to the growth observed in cultures of traditional type containing the aforesaid nutrients.

Therefore a further aspect of the present invention is a substrate comprising the aforesaid autologous type platelet gel, PPP gel or PRP gel on which cells can grow and proliferate in vivo.

Finally, a further aspect of the present invention is the use of the aforementioned autologous type platelet gel, PPP gel or PRP gel as promoter for the growth and proliferation of cells in vitro as a replacement for analogous heterologous type cell growth and proliferation promoters.

The aqueous solution which constitutes the activator of the present invention is different from that considered in patent application W001/43787 already mentioned, by the fact that it contains batroxobin in pure form and is devoid of phenols.

Therefore a further aspect of the present invention is the aforesaid aqueous solution.

DESCRIPTION OF THE FIGURES Figure 1 shows the platelet gel obtained with the activator of the present invention containing batroxobin at a concentration equal to 10 BU/ml.

Figure 2 shows PPP gel obtained with the activator of the present invention

containing batroxobin at a concentration equal to 10 BU/ml.

Figure 3A shows an enlarged (100x) inverted microscope photo of human primary gingival fibroblasts cultivated using conventional techniques and in the presence of FCS.

Figure 3B shows an enlarged (100x) photo of human primary gingival fibroblasts cultivated in the presence of platelet gel containing the activator in which batroxobin in pure form has a concentration equal to 0.5 BU/ml.

Figure 4A shows a block diagram relating to the number of HUVEC cells on the 1St and 5th days of culture obtained using gelatin under standard conditions as the growth medium (control) or using as the culture medium platelet gel, indicated by PC in the figure, and PPP gel, indicated by PPP in the figure, both said gels being prepared with the activator of the present invention containing batroxobin in a concentration equal to 0.5 BU/ml, and where the number of cells is shown by the y-axis.

Figure 4B shows a photo of HUVEC cells on the 5th day of culture grown under standard conditions in the presence of gelatin ; figure 4C shows a photo of the cells grown in the presence of platelet gel obtained with the activator of the present invention containing batroxobin at a concentration equal to 0.5 BU/ml, on the 5th day of culture; figure 4D shows a photo of the cells grown in the presence of PPP gel obtained with the activator of the present invention containing batroxobin at a concentration equal to 0.5 BU/ml, on the 5th day of culture.

DETAILED DESCRIPTION OF THE INVENTION The definition"essentially pure"means batroxobin with a degree of purity greater than 90%, while the definition"essentially devoid of phenols"indicates that the quantity of phenols in the aqueous solution of the present invention is less than 0. 01%, preferably 0. 001%.

The batroxobin used in the preparation of the aqueous solution of the present invention is preferably in lyophilised form.

According to a particular embodiment, the batroxobin used in the present invention is batroxobin moojeni sold by Pentapharm (Basel).

The aqueous solution of the present invention can contain other salts such as for

example sodium chloride or phosphates.

The calcium salts used in the activator of the present invention can be inorganic calcium salts and preferably calcium chloride, or organic salts and preferably calcium gluconate.

The calcium salt concentration is preferably between 5 x 10-5 and 0.5 M.

More preferably calcium gluconate is employed and even more preferably it is present in the aqueous solution of the invention in concentrations of between 0.1 and 0.2 M.

The batroxobin concentration in the aqueous solution of the present invention is preferably between 0.05 and 30, more preferably between 0.2 and 20, and even more preferably between 0.25 and 10 BU/ml The activator of the present invention can be used to induce the formation of platelet gel, PPP gel or PRP gel obtained from hemocomponents, such as plasma buffy coat, platelet concentrates, platelet rich plasma and platelet poor plasma.

Preferably, the platelet concentrate used in the preparation of platelet gel according to the method of the present invention is prepared by the method described in the above mentioned W001/43787, and which we incorporate in the present description as reference.

In particular, said method comprises the following steps: a) centrifuging the blood at 180 g for 20 minutes thereby obtaining 2 phases, the dark and heavier phase consisting of precipitated red and white blood cells and the clear and lighter phase consisting of PRP, b) centrifuging the PRP at 580 g for 15 minutes thereby obtaining 2 phases: a dense bottom or"pellet"of platelets and an upper phase consisting of PPP, c) recovering the pellet to which a minimum quantity of PPP is added to resuspend the platelets.

The PPP used in the process for preparing the corresponding gel is preferably obtained as described in the aforementioned patent application W001/43787, and virtually coincides with the aforestated process, the only difference being that in place of stage (c) this latter process proposes a stage (c') which, in particular, involves the recovery of PPP alone.

The PRP used in the process for preparing the corresponding gel is preferably

obtained as described in the aforementioned patent application W001/43787, and in practice comprises the aforementioned stage (a) of the aforesaid processes and a further stage (b') which consists of recovering PRP alone.

The cells able to grow and proliferate in vivo on a substrate comprising platelet gel, PPP gel or PRP gel of the present invention, or those able to grow and proliferate in vitro with only platelet gel, PPP gel or PRP gel of the present invention as cell growth and proliferation promoter, are preferably chosen from: fibroblasts, keratinocytes, osteoblasts, myocytes, nerve cells, cardiac muscle cells, endothelial cells and stem cells.

The present invention finds particular application in the field of cell grafting.

In this respect when a histological sample is taken, such as skin, bone, gingiva, muscle, to obtain the primary culture in vitro, a whole blood sample (40-50 ml) is also taken during the same session to be used for preparing the PC, PPP or PRP.

Thus both histological and whole blood samples arrive at the laboratory ready for processing. The PC and PPP can be conserved under agitation at 22 degrees for 5 days; or, alternatively, they, and preferably the PPP, can be frozen and then activated when the cells have to be seeded so as to eliminate the use of their culture medium or, certainly, heterologous serum.

The wound care activation kit according to the present invention comprises: A) a vial of calcium gluconate solution, B) a vial of lyophilised batroxobin, C) means for taking the content of the aforesaid two vials, D) means for taking respectively the autologous hemocomponent to be activated (PC, PPP and PRP respectively), E) at least one petri dish wherein said respectively platelet, PPP or PRP gel forms, said petri dish being provided with a lid having at least two openings with hermetical closure means, said openings being suitable for receiving respectively the means (C) and (D), The surgery activation kit further object of the present invention comprises the same components (A)- (D) of the wound care activation kit with the sole difference that in place of the aforementioned petri dish, namely the components (E), it encompasses the component (E'), consisting in at least one dappen useful to

collect the bone and for obtaining respectively the platelet, PPP and PRP gel, said dappen being provided with a lid having at least two openings with hermetical closure means, said openings being suitable for receiving respectively the means (C) and (D).

According to a preferred embodiment the means (C) and (D) in both the aforementioned kits consist of two syringes without needle.

According to a further preferred embodiment, the lid covering respectively the petri dish in the wound care activation kit according to the present invention and the dappen in the surgical activation kit is a screwing lid.

The two openings provided with hermetic closure present in said lid may be of any type known in the art. According to a particularly preferred embodiment and in case said means (C) and (D) are syringes without needles, said openings with hermetic closure consist of two padlocks suitable for receiving said syringes.

According to a further particularly preferred embodiment both kits further comprise a pair of pinches to take up the gel from the petri dish or from the dappen once it has formed.

According to a particularly preferred embodiment the kit contains a pipette allowing to take up the transudate once the gel has formed from the dappen or the petri dish.

This transudate may be used on its turn as an activator for a further gel precipitation by a further addition of a starting hemocomponent PC, PPP or PRP.

According to another preferred embodiment the wound care activation kit comprises two petri dishes, the first one may be used for precipitating the platelet gel, the remaining PPP obtained after separation of the pellets of platelets in the process for preparing the platelet gel from the PC may be added in said second petri dish in which after contemporaneous addition of batroxobin and calcium gluconate or of the aforementioned transudate the PPP gel forms.

According to a particularly preferred embodiment the method according to the present invention carried out in the kit may be accomplished as follows : i) the calcium gluconate solution is added by means of one of the aforesaid two syringes to the vial containing the lyophilised batroxobin, ii) the PC, PPP or PRP is taken by means of the aforesaid second syringe,

iii) the two syringes of step (i) and (ii) are screwed on the aforesaid two openings the lid of the Petri dish or dappen, iv) the piston of both syringes are contemporaneously pressed to allow the content of the syringes to flow into the petri dish or dappen, v) the mixture contained in the dappen is stirred with a rotating movement, vi) the lid of the petri dish or dappen is unscrewed and the platelet gel is recovered by means of the pinches, and the transudate remaining in the petri dish or dappen is recovered by means of the pipette.

By way of non-limiting illustration, some examples are given of preparing platelet gel and PPP gel obtained with the activator of the present invention and cytotoxicity and cell growth tests using platelet gel and PPP gel prepared using the activator of the present invention as growth and cell proliferation promoter.

EXAMPLE 1-Preparation of platelet and fibrin gel EXAMPLE 1 A Preparation of the activator The lyophilised form of batroxobin used was supplied by Pentapharm and consisted of batroxobin moojeni 500 NIH/ml-614 NIH/mg.

Lyophilised batroxobin is suspended in PBS (Dulbecco's Phosphate Buffer Saline 1x).

Batroxobin reconstituted in 1 ml of PBS has a concentration of 500 NIH/ml.

The aforesaid solution of batroxobin (500 NIH/ml) was further diluted, again with PBS, to obtain different concentrations, namely 0.5-1-5-10-20 NIH/ml.

The following activation mixtures were prepared: 1) 0.5 ml batrox 0.5 BU/ml + 0.5 ml of calcium gluconate (0. 23M), the final batroxobin concentration thus being 0.25 BU/ml, 2) 0.5 ml batrox 1 BU/ml + 0.5 ml of calcium gluconate (0.23M), the final batroxobin concentration thus being 0.5 BU/ml, 3) 0.5 mi batrox 5 BU/ml + 0.5 ml of calcium gluconate (0. 23M), the final batroxobin concentration thus being 2.5 BU/mi, 4) 0.5 ml batrox 10 BU/ml + 0.5 ml of calcium gluconate (0. 23M), the final batroxobin concentration thus being 5 BU/ml, 5) 0.5 ml batrox 20 BU/ml + 0.5 ml of calcium gluconate (0. 23M), the final batroxobin concentration thus being 10 BU/ml.

EXAMPLE 1-A'Preparation of the activator according to the method described in W001/43787.

1 ml of 0.23 M calcium gluconate is added to 1 ml botropase containing batroxobin at a concentration equal to 1 NIH/ml, so the final solution presents a batroxobin concentration equal to 0.5 NIH/ml EXAMPLE 1-B-Preparation of platelet gel The platelet concentrate was then prepared as described in W001/43787 to which the activators (1), (2) and (5) were added.

Activation times of PC, i. e. the formation times of the corresponding platelet gel, are given for each type of activator in Table 1.

Table 1 Activator (concentration) Activation times 1) 0. 25 BU/ml 5 minutes 2) 0.5 BU/ml 3 minutes 5) 10 BU/ml 30-40 seconds

EXAMPLE 1-B' The PC, prepared as described in W001/43787, is activated with the activator containing Botropase, whose preparation is described in example 1A'. Activation time, i. e. formation of the platelet gel, is about 7 minutes.

EXAMPLE 1 C-Precipitation of PPP gel PPP, prepared as described in W001/43787, is separated from the platelets precipitated in pellet form.

The platelet poor plasma is then activated with activators 1 and 5, whose preparation was described in example 1A. Activation times of the platelet poor plasma, i. e. the formation times of the corresponding platelet gel, are given for each type of activator in Table 2.

Table 2

Activator (concentration) Activation times 1) 0. 25 BU/ml 8-10 minutes 5) 10 BU/ml 2 minutes

EXAMPLE 1D' The PPP prepared as described in Example 1 C is activated with the activator containing botropase, whose preparation is described in Example 1A'. Activation time, namely the formation of the PPP gel is 15-20 minutes.

Analysis of the above data indicates that in the platelet gel formation, the activator according to the present invention with batroxobin having a concentration of 0.25 BU/ml corresponding to 0.045 NIH/ml, namely a concentration being of 1 magnitude order lower than that the formulation with botropase (0. 5NIH/ml) is lower (5minutes) than that obtained with botropase (7minutes).

The activation times are decidedly much shorter than those obtained with botropase by increasing the substantially pure batroxobin concentration to respectively to 0.5 BU (0. 09NIH/ml) (3 minutes) and to 10 BU/ml (2 NlH/ml) (30-40 seconds).

Almost the same consideration can be made when analysing the results obtained for PPP gel precipitation with the activator according to the present invention and those obtained with botropase, since by using a concentration of batroxobin of 1 magnitudo order lower than that used with botropase (0.25 BU/ml = 0.045 NIH/ml) the activation times are decidedly much lower (7minutes), than those obtained when using the activator containing botropase (15-20 minutes). The situation is even better when the activator according to the present invention contains batroxobin at higher concentration (10BU/ml = 1.8 NIH/ml) since the activation time is only 2 minutes.

EXAMPLE 2. Preparation of human gingival fibroblasts (HGF) The primary culture of human fibroblasts was obtained by a gingival biopsy of about 2x1x1 mm conducted under sterile conditions on a healthy patient, immediately added to a solution of PBS (Sigma) containing 10% penicillin/streptomycin (antibiotic) and transported to the laboratory.

The tissue was then finely sectioned. The pieces were washed in PBS then were transferred into Petri dishes containing 5 mg/ml of Dispase II, according to the procedure described in"Tissue Engineering Technology for Gingival Augmentation Procedures: A Case Report"Int. J. Periodontics Restorative Dent. 200; 20 553- 559. After incubation at 37°C for 1 hour in a 37°C incubator with 5% C02 and 98%

relative humidity, the epidermal layer was delicately removed and the dermis washed with PBS. The human gingival fibroblasts (HGF) were obtained by digestion of the dermis devoid of epidermis using a solution of 80 IU/ml type I collagenase. The cells grew in L-glutamine free MEM (Minimum Essential Eagle's medium), with 10% foetal bovine serum (FCS), streptomycin (100pg/l), penicillin (100 IU/ml) and 2 mmol of L-glutamine ; the medium was renewed twice a week.

To check the number of cells and their morphology, the cells were observed at various time intervals with an inverted microscope (Leica DM IL).

The human gingival fibroblasts used in the experiments were at the fourth passage.

Fibroblasts derived from cell line L929 stabilised in vitro were used in addition to human gingival fibroblasts.

EXAMPLE 3-CYTOTOXICITY TESTS EXAMPLE 3A-Cytotoxicity tests on platelet gel and PPP gel formed by treating with activator containing batroxobin as botropase. as proposed in W001/43787.

Platelet gel and PPP gel were obtained using the activator prepared as described in example 1A'. This activator contains batroxobin (as botropase) in a concentration equal to 0.5 NIH/ml. Another activator was prepared, characterised by the final concentration of batroxobin (as botropase) being 0.25 NIH/ml. Human fibroblasts which were isolated, cultivated and proliferated as described in example 2A were used for these experiments.

Cytotoxicity was evaluated using cell lines obtained as described in example 2.

The culture medium used for the experiment was L-glutamine free Minimum Essential Eagle's Medium (MEM) (Sigma, Milan), with 10% foetal bovine serum (FCS), streptomycin (100pg/I), penicillin (100 IU/ml) and 2 mmol L-glutamine all in a 250 ml Falcon@ plastic cell culture flask. The cells were cultivated in an incubator at 37°C, 5% C02 and 98% relative humidity; they were then harvested before reaching confluence using a sterile trypsin solution, resuspended in cell culture medium and diluted therein until cell concentration was equal to about 1 x105 cells/ml.

3.5 ml of said cell suspension were added to Falcon@ polystyrene six-well cell culture plates (9.6 cm2 growing area), containing the aforesaid platelet gels formed

using the aforesaid activators with the aforesaid concentrations of batroxobin as botropase.

By 24 hours the cells which were seeded with botropase 0.5 NIH/ml were distressed with respect to controls, and even more so after 72 hours. The control cells presented normal morphology and were thriving. The cells which were plated with 0.25 NIH/ml botropase were thriving at 24 hours after seeding and at 72 hours presented not only totally normal morphology but, surprisingly, were definitely in greater numbers than the control.

EXAMPLE 3B-Cytotoxicity tests with platelet gel and PPP gel obtained by using as activator the aqueous solutions of the present invention.

Platelet gels were prepared with the activators of the present invention comprising aqueous solutions containing calcium gluconate and lyophilised batroxobin reconstituted in PBS in respective concentrations of 0.5-1-10-20 BU/ml, prepared as described in example 1 A.

Cytotoxicity was evaluated using cell lines obtained as described in example 2 and prepared for the cytotoxicity test as described in the preceding example 3A, and subsequently seeded as described in the aforesaid example with the only difference being that the cell culture seeded contain platelet gels formed by using the aforesaid activators containing the aforesaid concentrations of pure batroxobin.

After 3 days'growth in incubators at 37°C, 5% COa and 98% relative humidity, any cell death and cell morphology were verified. The cell monolayer was observed with an inverted microscope (DM IL Leica) to evaluate cell death or reduction in cell number. Morphology was also carefully checked and compared with the negative control, i. e. cells grown in the absence of the aforesaid platelet gels.

No cell death was detected by inverted microscope for any of the cells grown with platelet gels obtained with different concentrations of batroxobin. Even the platelet gel containing 20 BU/ml of batroxobin did not inhibit cell growth.

After 24 hours cell morphology was shown to be totally normal and completely comparable to the control and even after 72 hours no cytotoxic effect was verified.

Therefore it can be concluded that pure lyophilised batroxobin does not appear to be cytotoxic even at concentrations higher than 20 BU/ml.

EXAMPLE 4 cell growth of human gingival fibroblasts (HGF) in the presence of platelet gels obtained by using aqueous solutions of pure batroxobin as activator.

HGF cells were isolated, cultivated and proliferated as described in example 2 and subsequently prepared as described in experiment 3A, using 3.5 ml of a cell suspension in MEM medium without foetal bovine serum (1x105 cells/ml), obtained by means of a sterile trypsin/EDTA solution. This cell suspension was seeded into Falcon@ polystyrene six-well cell culture plates (9.6 cm2 growing area) containing platelet gel formed with the activators of to the present invention, comprising aqueous solutions containing calcium gluconate and lyophilised batroxobin reconstituted in PBS in respective concentrations of 0.5-1-10 BU/ml, prepared as described in example 1A.

After 3 days'growth in an incubator at 37°C, 5% C02 and 98% relative humidity, cell death, cell numbers and cell morphology were evaluated. The cell monolayer was analysed with an inverted microscope (DM IL, LEICA). The cells were carefully checked and compared with the results obtained from the negative control i. e. cells grown in MEM containing 10% foetal bovine serum in the polystyrene cell culture plates.

It was concluded, particularly evident from photos 1A and 1B, that all the wells containing platelet gel prepared with the activator showed a considerable increase in cell density compared with the corresponding control.

EXAMPLE 5-growth of endothelial HUVEC cells in the presence of platelet gel and PPP gel formed with the activator of the present invention.

A culture medium with the following components is prepared: 2% gelatin, TC-199 cell culture medium, 20% FCS (foetal calf serum), 1% RDGF (retinal-derived growth factor), 1 % nutridoma and 1.2 ml heparin per 500 mi culture medium.

The HUVEC cells are then seeded directly onto the gelatin. The cells then grow quickly in an incubator under standard conditions: 5% C02, 37°C temperature and 98% relative humidity. The control was prepared in this manner.

To verify the ability of platelet gel and PPP gel to enhance cell growth, the PPP and platelet gels of the present invention were prepared using an activator of the present invention containing batroxobin at concentrations equal to 0.5 BU/ml ; on these were seeded the cells with neither their medium nor the constituents

(nutridoma, RDGF, FCS, heparin) necessary for the control. On the first and fifth day of culturing, the control cells and the cells grown in platelet gel and PPP gel were counted. The data obtained are reported in table 3 and Figure 4 A-4B-4C- 4D.

Table 3 Gelatin (control) Platelet gel PPP gel 1 S day 50, 000 50, 000 50, 000 5th day 80, 000 120, 000 100, 000